Malaria eradication is a global health priority that will require interventions on multiple scales. The sporozoite transmission stages infect the liver and are important targets for intervention as very few parasites successfully initiate infection of the mammalian host. Early stages of infection have typically been studied using the rodent models of malaria, and the search for infectivity-associated genes has been restricted to candidate-based approaches. The recent development of liver-chimeric humanized mouse models has made a dramatic impact on the field, now enabling sporozoite transmission and liver stage studies of Plasmodium falciparum and the implementation of genetic crosses. Therefore we propose to utilize a genetic cross, conducted in the liver-chimeric humanized mouse (FRG huHep), to create a recombinant mapping population of progeny parasites that have varying levels of sporozoite infectivity. This parasite population can be used to characterize the transcriptional regulatory network underlying infectivity using systems genetics. Our preliminary data show that two clonal recombinant progeny from a genetic cross, exhibited higher sporozoite infectivity levels than the parent NF54 laboratory strain. We will characterize the remaining parasites from this cross to determine the clonal line that is most infectious, and use this parasite to perform a back cross in the FRG huHep mouse. We will identify genetic variation associated with the measured phenotypes using quantitative trait loci (QTL) mapping. Because QTL mapping will identify large linkage blocks of DNA that contain several dozen genes that may be associated with infectivity, we will then use expression QTL (eQTL) mapping, co-expression network analysis and Bayesian network construction to systematically describe the transcriptional network regulating infectivity. This study will provide genetic and transcriptional analysis of P. falciparum sporozoites on an unprecedented level, defining the molecular processes that govern sporozoite infectivity on a global scale. Dissection of the sporozoite-infectivity associated pathways and regulatory networks can be used to identify drug and vaccine targets which specifically intervene against sporozoite infection.